Bacillus thuringiensis toxins with improved activity

ABSTRACT

The subject invention pertains to  B.t.  toxins active against pests. More specifically, the subject invention pertains to truncated Cry6A toxins. These activated toxins are particularly effective for controlling coleopteran pests such as the corn rootworm and the alfalfa weevil.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This is a continuation of U.S. patent application Ser. No.09/222,594, filed Dec. 28, 1998, which is a divisional of U.S. patentapplication Ser. No. 08/904,278, filed Jul. 31, 1997, now U.S. Pat. No.5,874,288.

BACKGROUND OF THE INVENTION

[0002] Insects and other pests cost farmers billions of dollars annuallyin crop losses and in the expense of keeping these pests under control.The losses caused by pests in agricultural production environmentsinclude decrease in crop yield, reduced crop quality, and increasedharvesting costs.

[0003] Coleopterans are an important group of agricultural pests whichcause a very large amount of damage each year. Examples of coleopteranpests include alfalfa weevils and corn rootworm.

[0004] The alfalfa weevil, Hypera postica, and the closely relatedEgyptian alfalfa weevil, Hypera brunneipennis, are the most importantinsect pests of alfalfa grown in the United States, with 2.9 millionacres infested in 1984. An annual sum of 20 million dollars is spent tocontrol these pests. The Egyptian alfalfa weevil is the predominantspecies in the southwestern U.S., where it undergoes aestivation (i.e.,hibernation) during the hot summer months. In all other respects, it isidentical to the alfalfa weevil, which predominates throughout the restof the U.S.

[0005] The larval stage is the most damaging in the weevil life cycle.By feeding at the alfalfa plant's growing tips, the larvae causeskeletonization of leaves, stunting, reduced plant growth, and,ultimately, reductions in yield. Severe infestations can ruin an entirecutting of hay. The adults, also foliar feeders, cause additional, butless significant, damage.

[0006] Approximately 9.3 million acres of U.S. corn are infested withcorn rootworm species complex each year. The corn rootworm speciescomplex includes the northern corn rootworm, Diabrotica barberi, thesouthern corn rootworm, D. undecimpunctata howardi, and the western cornrootworm, D. virgifera virgifera. The soil-dwelling larvae of theseDiabrotica species feed on the root of the corn plant, causing lodging.Lodging eventually reduces corn yield and often results in death of theplant. By feeding on cornsilks, the adult beetles reduce pollinationand, therefore, detrimentally effect the yield of corn per plant. Inaddition, members of the genus Diabrotica attack cucurbit crops(cucumbers, melons, squash, etc.) and many vegetable and field crops incommercial production as well as those being grown in home gardens.

[0007] Control of corn rootworm has been partially addressed bycultivation methods, such as crop rotation and the application of highphosphate levels to stimulate the growth of an adventitious root system.In addition, an emerging two-year diapause (or overwintering) trait ofNorthern corn rootworms is disrupting crop rotation in some areas.However, chemical insecticides are relied upon most heavily to guaranteethe desired level of control. Insecticides are either banded onto orincorporated into the soil. The major problem associated with the use ofchemical insecticides is the development of resistance among the treatedinsect populations.

[0008] Over $250 million worth of insecticides are applied annually tocontrol corn rootworms alone in the United States. Even with insecticideuse, rootworms cause over $750 million worth of crop damage each year,making them the most serious corn insect pest in the Midwest.

[0009] Damage to plants caused by nematodes is also a prevalent andserious economic problem. Nematodes cause wide-spread and serious damagein many plant species. Many genera of nematodes are known to cause suchdamage. Plant-parasitic nematodes include members of the PhylumNematoda, Orders Tylenchida and Dorylaimide. In the Order Tylenchida,the plant-parasitic nematodes are found in two Super Families:Tylenchoidea and Criconematoidea.

[0010] There are more than 100,000 described species of nematodes.

[0011] Chemical pesticides have provided an effective method of pestcontrol; however, the public has become concerned about the amount ofresidual chemicals that might be found in food, ground water, and theenvironment. Stringent new restrictions on the use of pesticides and theelimination of some effective pesticides form the marketplace couldlimit economical and effective options for controlling costly pests.Thus, there is an urgent need to identify pest control methods andcompositions which are not harmful to the environment.

[0012] Nematicides routinely used for control of plant-parasiticnematodes are rapidly being pulled from the market as concern forenvironmental safety increases. In the year 2001, Methyl Bromide, amainstay in the control of such parasites, will no longer be marketed inthe United States. Therefore, less harmful control agents are clearlyneeded.

[0013] The use of chemical pesticides to control corn rootworm and othercoleopteran pests, as well as nematodes, has several drawbacks.Pesticide use often raises environmental concerns such as contaminationof soil and of both surface and underground water supplies. Working withpesticides may also pose hazards to the persons applying them.

[0014] The regular use of chemical pesticides for the control ofunwanted organisms can select for chemical resistant strains. Chemicalresistance occurs in many species of economically important insects andhas also occurred in nematodes of sheep, goats, and horses. The regularuse of chemical toxins to control unwanted organisms can select fordrug-resistant strains. This has occurred in many species ofeconomically important insects and has also occurred in nematodes ofsheep, goats, and horses. For example, an accepted methodology forcontrol of nematodes has centered around the drug benzimidazole and itscongeners. The use of these drugs on a wide scale has led to manyinstances of resistance among nematode populations (Prichard, R. K. etal. [1980] “The problem of anthelmintic resistance in nematodes,” Austr.Vet. J 56:239-251; Coles, G. C. [1986] “Anthelmintic resistance insheep,” In Veterinary Clinics of North America: Food Animal Practice,Vol 2:423-432 [Herd, R. P., eds.] W. B. Saunders, New York). There aremore than 100,000 described species of nematodes. The development ofpesticide resistance necessitates a continuing search for new controlagents having different modes of action.

[0015] At the present time there is a need to have more effective meansto control the many coleopterans and nematodes that cause considerabledamage to susceptible hosts and crops. Advantageously, such effectivemeans would employ specific biological agents.

[0016] The soil microbe Bacillus thuringiensis (B.t.) is aGram-positive, spore-forming bacterium characterized by parasporalcrystalline protein inclusions. These inclusions often appearmicroscopically as distinctively shaped crystals. The proteins can behighly toxic to pests and specific in their toxic activity. Certain B.t.toxin genes have been isolated and sequenced, and recombinant DNA-basedB.t. products have been produced and approved for use. In addition, withthe use of genetic engineering techniques, new approaches for deliveringthese B.t. endotoxins to agricultural environments are underdevelopment, including the use of plants genetically engineered withendotoxin genes for insect resistance and the use of stabilized intactmicrobial cells as B.t. endotoxin delivery vehicles (Gaertner, F. H., L.Kim [1988] TIBTECH 6:54-57). Thus, isolated B.t. endotoxin genes arebecoming commercially valuable.

[0017] Until fairly recently, commercial use of B.t. pesticides has beenlargely restricted to a narrow range of lepidopteran (caterpillar)pests. Preparations of the spores and crystals of B. thuringiensissubsp. kurstaki have been used for many years as commercial insecticidesfor lepidopteran pests. For example, B. thuringiensis var. kurstaki HD-1produces a crystalline δ-endotoxin which is toxic to the larvae of anumber of lepidopteran insects.

[0018] In recent years, however, investigators have discovered B.t.pesticides with specificities for a much broader range of pests. Forexample, other species of B.t., namely israelensis and morrisoni (a.k.a.tenebrionis, a.k.a. B.t. M-7, a.k.a. B.t. san diego), have been usedcommercially to control insects of the orders Diptera and Coleoptera,respectively (Gaertner, F. H. [1989] “Cellular Delivery Systems forInsecticidal Proteins: Living and Non-Living Microorganisms,” inControlled Delivery of Crop Protection Agents, R. M. Wilkins, ed.,Taylor and Francis, New York and London, 1990, pp. 245-255.). See alsoCouch, T. L. (1980) “Mosquito Pathogenicity of Bacillus thuringiensisvar. israelensis,” Developments in Industrial Microbiology 22:61-76; andBeegle, C. C., (1978) “Use of Entomogenous Bacteria in Agroecosystems,”Developments in Industrial Microbiology 20:97-104. Krieg, A., A. M.Huger, G. A. Langenbruch, W. Schnetter (1983) Z. ang. Ent. 96:500-508,describe Bacillus thuringiensis var. tenebrionis, which is reportedlyactive against two beetles in the order Coleoptera. These are theColorado potato beetle, Leptinotarsa decemlineata, and Agelastica alni.

[0019] More recently, new subspecies of B.t. have been identified, andgenes responsible for active δ-endotoxin proteins have been isolated(Höfte, H., H. R. Whiteley [1989] Microbiological Reviews52(2):242-255). Höfte and Whiteley classified B.t. crystal protein genesinto four major classes. The classes were CryI (Lepidoptera-specific),CryII (Lepidoptera- and Diptera-specific), CryIII (Coleoptera-specific),and CryIV (Diptera-specific). The discovery of strains specificallytoxic to other pests has been reported (Feitelson, J. S., J. Payne, L.Kim [1992] Bio/Technology 10:271-275). CryV was proposed to designate aclass of toxin genes that are nematode-specific.

[0020] Other classes of B.t. genes have now been identified.

[0021] The 1989 nomenclature and classification scheme of Höfte andWhiteley for crystal proteins was based on both the deduced amino acidsequence and the host range of the toxin. That system was adapted tocover 14 different types of toxin genes which were divided into fivemajor classes. As more toxin genes were discovered, that system startedto become unworkable, as genes with similar sequences were found to havesignificantly different insecticidal specificities. The number ofsequenced Bacillus thuringiensis crystal protein genes currently standsat about 50. A revised nomenclature scheme has been proposed which isbased solely on amino acid identity (Crickmore et al. [1996] Society forInvertebrate Pathology, 29th Annual Meeting, IIIrd InternationalColloquium on Bacillus thuringiensis, University of Cordoba, Cordoba,Spain, September 1-6, abstract). The mnemonic “cry” has been retainedfor all of the toxin genes except cytA and cytB, which remain a separateclass. Roman numerals have been exchanged for Arabic numerals in theprimary rank, and the parentheses in the tertiary rank have beenremoved. Many of the original names have been retained, with the notedexceptions, although a number have been reclassified.

[0022] The cloning and expression of a B.t. crystal protein gene inEscherichia coli has been described in the published literature(Schnepf, H. E., H. R. Whiteley (1981) Proc. Natl. Acad. Sci. USA78:2893-2897). U.S. Pat. No. 4,448,885 and U.S. Pat. No. 4,467,036 bothdisclose the expression of B.t. crystal protein in E. coli. U.S. Pat.Nos. 4,990,332; 5,039,523; 5,126,133; 5,164,180; 5,169,629; and5,286,485 are among those which disclose B.t. toxins having activityagainst lepidopterans. U.S. Pat. Nos. 4,797,276 and 4,853,331 discloseB. thuringiensis strain tenebrionis which can be used to controlcoleopteran pests in various environments. U.S. Pat. No. No. 4,918,006discloses B.t. toxins having activity against dipterans.

[0023] A small number of research articles have been published about theeffects of delta endotoxins from B. thuringiensis species on theviability of nematode eggs. Bottjer, Bone and Gill, ([1985] ExperimentalParasitology 60:239-244) have reported that B.t. kurstaki and B.t.israelensis were toxic in vitro to eggs of the nematode Trichostrongyluscolubriformis. In addition, 28 other B.t. strains were tested withwidely variable toxicities. Ignoffo and Dropkin ([1977] J. Kans.Entomol. Soc. 50:394-398) have reported that the thermostable toxin fromBacillus thuringiensis (beta exotoxin) was active against a free-livingnematode, Panagrellus redivivus (Goodey); a plant-parasitic nematode,Meloidogyne incognita (Chitwood); and a fungus-feeding nematode,Aphelenchus avena (Bastien). Beta exotoxin is a generalized cytotoxicagent with little or no specificity. Also, Ciordia and Bizzell ([1961]Jour. of Parasitology 47:41 [abstract]) gave a preliminary report on theeffects of B. thuringiensis on some cattle nematodes.

[0024] U.S. Pat. No. 5,151,363 and U.S. Pat. No. 4,948,734 disclosecertain isolates of B.t. which have activity against nematodes. OtherU.S. Pat. Nos. which disclose activity against nematodes include5,093,120; 5,236,843; 5,262,399; 5,270,448; 5,281,530; 5,322,932;5,350,577; 5,426,049; and 5,439,881. As a result of extensive researchand investment of resources, other patents have issued for new B.t.isolates and new uses of B.t. isolates. See Feitelson et al., supra, fora review. However, the discovery of new B.t. isolates and new uses ofknown B.t. isolates remains an empirical, unpredictable art.

[0025] Some Bacillus thuringiensis toxins which are active against cornrootworm and other a coleopterans are now known. For example, U.S. Pat.No. 4,849,217 discloses various isolates, including PS52A1 and PS86A1,as having activity against alfalfa weevils. U.S. Pat. No. 5,208,017discloses PS86A1 as a having activity against Western corn rootworm.U.S. Pat. No. 5,427,786 and 5,186,934 each disclose B.t. isolates andtoxins active against coleopterans. Specifically disclosed in thesepatents is the isolate known as PS86A1 and a coleopteran-active toxinobtainable therefrom known as 86A1. Toxin 86A1 is now also known asCry6A (CryVIA). The wild-type Cry6A toxin is about 54-58 kDa.

[0026] A Cry6B toxin is also known. This toxin can be obtained from thePS69D1 isolate. The full length Cry6A and Cry6B toxins are known to haveactivity against nematodes. The following U.S. Pat. Nos. disclose, inpart, the PS69D 1 isolate as having activity against nematodes:4,948,734; 5,093,120; 5,262,399; and 5,439,881.

[0027] A generic formula for the amino acid sequence of CryVI toxins hasbeen disclosed in WO 92/19739, which also teaches that the full lengthtoxin has activity against nematodes. The PS52A1 and PS69D1 isolates aredisclosed therein. U.S. Pat. Nos. 5,262,159 and 5,468,636 also disclosea generic formula for toxins having activity against aphids.

[0028] Although the Cry6A toxin was known to inhibit the growth ofcertain coleopterans, it was not previously known that this toxin couldbe activated by truncation to yield a toxin that is lethal tocoleopterans, such as the western corn rootworm. In addition, there wasno suggestion that the truncated Cry6A would be active againstnematodes.

[0029] Some previous examples of truncations to other B.t. toxins areknown in the art. For example, the P2 (Cry2) toxins (Nicholles, E. N.,W. Ahmad, D. J. Ellar [1989] J. Bact. 171:5141-5147) exist as 61-63 kDaproteins. Proteolysis trims about 5 kDa off, leaving 56-58 kDa proteins.However, toxicity either remained unchanged or was worse by a factor of10. Furthermore, these proteins share no significant homology with Cry6toxins. Other articles which address certain aspects of the activityand/or function of portions of B.t. toxins include Adang, M. J., M. J.Staver, T. A. Rocheleau et al. (1985) Gene 36:289-300; Wabiko, H., K. C.Raymond, L. A. Bulla, Jr. (1986) DNA 5:305-314 (Medline 863000920);Schnepf, H. E, H. R. Whiteley (1985) J. Biol. Chem. 260:6273-6280; U.S.Pat. No. 5,468,636; U.S. Pat. No. 5,236,843; and EP 0462721.

[0030] The use of truncation to obtain activated Cry6A toxins asdescribed below is completely new to the B.t. art.

BRIEF SUMMARY OF THE INVENTION

[0031] The subject invention concerns materials and methods useful inthe control of pests and, particularly, plant pests. Specifically, thesubject invention provides new truncated B.t. toxins useful for thecontrol of coleopteran pests, including corn rootworm. The subjectinvention further provides toxins useful for the control of nematodes.The subject invention further provides nucleotide sequences which encodethese toxins.

[0032] In a preferred embodiment of the subject invention, truncatedforms of cry6A B.t. toxins have been found to be particularly activeagainst corn rootworm. Truncated toxins described herein can also beused to control nematodes. Specifically exemplified herein is atruncated cry6A toxin which has amino acids removed from the N-terminusand the C-terminus and is about 40-50 kDa. In a preferred embodiment,the toxins of the subject invention are produced by genes which encodethe highly active truncated proteins. As described herein, the truncatedtoxins of the subject invention can also be obtained through treatmentof B.t. culture supernatants and/or by growing B.t. cultures underappropriate conditions to result in the production of active toxins as aresult of the advantageous effects of proteases. Similarly, proteinexpressed by a recombinant host may be treated to obtain the truncatedtoxin.

[0033] In a preferred embodiment, the genes described herein whichencode pesticidal toxins are used to transform plants in order to conferpest resistance upon said plants. Such transformation of plants can beaccomplished using techniques well known to those skilled in the art andwould typically involve modification of the gene to optimize expressionof the truncated toxin in plants.

[0034] The subject invention also concerns the use of all or part of thetruncated toxins and genes in the production of fusion proteins andfusion genes.

BRIEF DESCRIPTION OF THE DRAWING

[0035]FIG. 1 is an amino acid by amino acid comparison of 86A1 and 69D1.

BRIEF DESCRIPTION OF THE SEQUENCES

[0036] SEQ ID NO. 1 is the nucleotide sequence of a full-length cry6Agene.

[0037] SEQ ID NO. 2 is the amino acid sequence of a full-length Cry6Atoxin.

[0038] SEQ ID NO. 3 is a full-length plant-optimized gene sequence forcry6A/86A1 gene.

[0039] SEQ ID NO. 4 is the full-length amino acid sequence encoded bySEQ ID NO. 3.

[0040] SEQ ID NO. 5 is the R443 truncated gene sequence.

[0041] SEQ ID NO. 6 is the amino acid sequence encoded by SEQ ID NO. 5.

[0042] SEQ ID NO. 7 is the truncated plant-optimized R390 gene sequence.

[0043] SEQ ID NO. 8 is the truncated protein sequence encoded by SEQ IDNO. 7.

[0044] SEQ ID NO. 9 is the nucleotide sequence of a full lengthcry6B/69D1 gene.

[0045] SEQ ID NO. 10 is the amino acid sequence of a full lengthCry6B/69D1 toxin.

DETAILED DISCLOSURE OF THE INVENTION

[0046] The subject invention concerns materials and methods for thecontrol of pests. In specific embodiments, the subject inventionpertains to truncated B.t. toxins having activity against coleopteranpests. The subject invention also provides toxins useful in the controlof nematodes.

[0047] The subject invention further concerns novel genes which encodethese pesticidal toxins. In a particularly preferred embodiment, thematerials and methods described herein can be used to control cornrootworm.

[0048] Isolates useful according to the subject invention are availableto those skilled in the art by virtue of deposits described in variousU.S. Pat. Nos., including U.S. Pat. No. 5,427,786; 5,186,934; and5,273,746. See also PCT international application number WO 93/04587.The PS86A1 (NRRL B-18400, deposited Aug. 16, 1988) and MR506 microbesare disclosed in these patents. The PS69D1 isolate (NRRL B-18247,deposited Jul. 28, 1987) has been disclosed in various U.S. Pat. Nos.including: 4,948,734; 5,093,120; 5,262,399; and 5,439,881.

[0049] The B.t. PS86A1 isolate produces an approximately 55 kDa toxin isreferred to as the 86A1 or 86A1(a) toxin. This toxin is a Cry6A toxin.The gene encoding this toxin has been cloned into Bacillus thuringiensisisolate MR506, which also expresses the Cry6A toxin.

[0050] In a preferred embodiment of the subject invention, theapproximately 55 kDa Cry6A toxin expressed by B.t. isolates PS86A1 andMR506 is truncated to yield a toxin of approximately 45 kDa having highbiological activity against coleopterans. This type of truncated toxinis referred to herein as the truncated Cry6A toxin. Advantageously, thistruncated toxin has been found to be particularly active against cornrootworm. This truncated toxin preferably has amino acids removed fromthe N-terminus and the C-terminus to yield the active truncated forms.However, truncated, activated toxins according to the subject inventionmay also be obtained by removing amino acids from either the N-terminus,only, or the C-terminus, only.

[0051] As described herein, the removal of amino acids to yield thetruncated active form can be accomplished using a variety of techniques.In a preferred embodiment, the gene is modified to encode the activetruncated form of the toxin. Alternatively, the truncated toxins of thesubject invention can be obtained by treatment of B.t. cultures orgrowing the cultures under appropriate conditions such that endogenousproteases cleave the protein to its highly active form.

[0052] The removal of portions of the N-terminus and the C-terminus ofthe 86A1 55 kDa toxin was found to result in an advantageous activationof this toxin which increased the potency of its activity. Removal ofamino acids can be accomplished by treatment with trypsin, preferably,or with another appropriate enzyme, or enzyme mixture, such as Pronase,chymotrypsin, or endogenous proteases in B.t. culture broths. Trypsinconcentration, time of incubation, and temperature are interdependentconditions and could be varied by a person skilled in the art to obtainthe desired final product of the digestion. Other biologically activefragments can be obtained by those skilled in the art using theteachings provided herein. Fragments having amino and/or carboxyltermini similar to that identified above would also show improvedinsecticidal activity.

[0053] As those skilled in the art having the benefit of this disclosurewould readily recognize, the specific media used to grow the B.t.culture can be modified to achieve optimum activation of the B.t. toxin.For example, the cell density can be modulated by adjusting or changingthe culture medium. Also, media having proteases therein can be used toenhance the activation of the B.t. toxins.

[0054] The full length B.t. toxins of about 55 kDa can be expressed andthen converted to highly active forms through addition of appropriatereagents and/or by growing the cultures under conditions which result inthe truncation of the proteins through the fortuitous action ofendogenous proteases. In an alternative embodiment, the full lengthtoxin may undergo other modifications to yield the active form of thetoxin. Adjustment of the solubilization of the toxin, as well as otherreaction conditions, such as pH, ionic strength, or redox potential, canbe used to effect the desired modification of the full length toxin toyield an active form.

[0055] One recombinant host which can be used to obtain the truncatedtoxin of the subject invention is MR506. The truncated toxin of thesubject invention can be obtained by treating the crystallineδ-endotoxin of Bacillus thuringiensis strain MR506 with a serineprotease such as bovine trypsin at an alkaline pH and preferably in theabsence of β-mercaptoethanol.

[0056] The subject invention also concerns the use of all or part of thetruncated toxins and genes in the production of fusion proteins andfusion genes.

[0057] Genes and toxins. The genes and toxins useful according to thesubject invention include not only the sequences specificallyexemplified but also shorter sequences, and variants, mutants, andfusion proteins which retain the characteristic pesticidal activity ofthe toxins specifically exemplified herein. As used herein, the terms“variants” or “variations” of genes refer to nucleotide sequences whichencode the same toxins or which encode equivalent toxins havingpesticidal activity. As used herein, the term “equivalent toxins” refersto toxins having the same or essentially the same biological activityagainst the target pests as the exemplified toxins.

[0058] It should be apparent to a person skilled in this art that genesencoding active toxins can be identified and obtained through severalmeans. The specific genes exemplified herein may be obtained from theisolates deposited at a culture depository as described above. Thesegenes, or portions or variants thereof, may also be constructedsynthetically, for example, by use of a gene synthesizer. Variations ofgenes may be readily constructed using standard techniques for makingpoint mutations. Also, fragments of these genes can be made usingcommercially available exonucleases or endonucleases according tostandard procedures. For example, enzymes such as Bal31 or site-directedmutagenesis can be used to systematically cut off nucleotides from theends of these genes. Also, genes which encode active fragments may beobtained using a variety of restriction enzymes. Proteases may be usedto directly obtain active fragments of these toxins.

[0059] Equivalent toxins and/or genes encoding these equivalent toxinscan be derived from B.t. isolates and/or DNA libraries using theteachings provided herein. There are a number of methods for obtainingthe pesticidal toxins of the instant invention. For example, antibodiesto the pesticidal toxins disclosed and claimed herein can be used toidentify and isolate other toxins from a mixture of proteins.Specifically, antibodies may be raised to the portions of the toxinswhich are most constant and most distinct from other B.t. toxins. Theseantibodies can then be used to specifically identify equivalent toxinswith the characteristic activity by immunoprecipitation, enzyme linkedimmunosorbent assay (ELISA), or Western blotting. Antibodies to thetoxins disclosed herein, or to equivalent toxins, or fragments of thesetoxins, can readily be prepared using standard procedures in this art.The genes which encode these toxins can then be obtained from themicroorganism.

[0060] Fragments and equivalents which retain the pesticidal activity ofthe exemplified toxins would be within the scope of the subjectinvention. Also, because of the redundancy of the genetic code, avariety of different DNA sequences can encode the amino acid sequencesdisclosed herein. It is well within the skill of a person trained in theart to create these alternative DNA sequences encoding the same, oressentially the same, toxins. These variant DNA sequences are within thescope of the subject invention. As used herein, reference to“essentially the same” sequence refers to sequences which have aminoacid substitutions, deletions, additions, or insertions which do notmaterially affect pesticidal activity. Fragments retaining pesticidalactivity are also included in this definition.

[0061] A further method for identifying the toxins and genes of thesubject invention is through the use of oligonucleotide probes. Theseprobes are detectable nucleotide sequences. Probes provide a rapidmethod for identifying toxin-encoding genes of the subject invention.The nucleotide segments which are used as probes according to theinvention can be synthesized using a DNA or RNA synthesizer and standardprocedures. The probe will normally have at least about 10 bases, moreusually at least about 18 bases, and may have up to about 50 bases ormore, usually not having more than about 200 bases if the probe is madesynthetically. However, longer probes can readily be utilized, and suchprobes can be, for example, several kilobases in length. The probesequence is designed to be at least substantially complementary to agene encoding a toxin of interest. The probe need not have perfectcomplementarity to the sequence to which it hybridizes. The probes maybe labelled utilizing techniques which are well known to those skilledin this art. Such a probe analysis provides a rapid method foridentifying potentially commercially valuable insecticidal endotoxingenes within the multifarious subspecies of B.t.

[0062] Various degrees of stringency of hybridization can be employed.The more stringent the conditions, the greater the complementarity thatis required for duplex formation. Severity can be controlled bytemperature, probe concentration, probe length, ionic strength, time,and the like. Preferably, hybridization is conducted under stringentconditions by techniques well known in the art, as described, forexample, in Keller, G. H., M. M. Manak (1987) DNA Probes, StocktonPress, New York, NY., pp. 169-170. Low-stringency hybridization is thepreferred method when a larger gene fragment is used.

[0063] As used herein “stringent” conditions for hybridization refers toconditions which achieve the same, or about the same, degree ofspecificity of hybridization as the conditions employed by the currentapplicants. Specifically, hybridization of immobilized DNA on Southernblots with 32P-labeled gene-specific probes was performed by standardmethods (Maniatis, T., E. F. Fritsch, J. Sambrook [1982] MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.). In general, hybridization and subsequent washes werecarried out under stringent conditions that allowed for detection oftarget sequences with homology to the exemplified toxin genes. Fordouble-stranded DNA gene probes, hybridization was carried out overnightat 20-25° C. below the melting temperature (Tm) of the DNA hybrid in 6XSSPE, 5X Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. Themelting temperature is described by the following formula (Beltz, G. A.,K. A. Jacobs, T. H. Eickbush, P. T. Cherbas, and F. C. Kafatos [1983]Methods of Enzymology, R. Wu, L. Grossman and K. Moldave [eds.] AcademicPress, New York 100:266-285).

[0064] Tm=81.5° C.+16.6 Log[Na+]+0.41(% G+C)-0.61(%formamide)-600/length of duplex in base pairs.

[0065] Washes are typically carried out as follows:

[0066] (1) Twice at room temperature for 15 minutes in 1X SSPE, 0.1% SDS(low stringency wash).

[0067] (2) Once at Tm−20° C. for 15 minutes in 0.2X SSPE, 0.1% SDS(moderate stringency wash).

[0068] For oligonucleotide probes, hybridization was carried outovernight at 10-20° C. below the melting temperature (Tm) of the hybridin 6X SSPE, 5X Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA.Tm for oligonucleotide probes can be determined by the“nearest-neighbor” method. See Breslauer et al., “Predicting DNA duplexstability from the base sequence,” Proc. Natl. Acad. Sci. USA, 83 (11):3746-3750 (June 1986); Rychlik and Rhoads, “A computer program forchoosing optimal oligonucleotides for filter hybridization, sequencingand in vitro amplification of DNA,” Nucleic Acids Res., 17 (21):8543-8551 (Nov. 11, 1989); Santa Lucia et al., “Improvednearest-neighbor parameters for predicting DNA duplex stability,”Biochemistry 35 (11): 3555-3562 (Mar. 19, 1996); Doktycz et al.,“Optical melting of 128 octamer DNA duplexes. Effects of base pairlocation and nearest neighbors on thermal stability,” J. Biol. Chem.,270 (15): 8439-8445 (Apr. 14, 1995). Alternatively, the Tm can bedetermined by the following formula:

[0069] Tm (° C.)=2(number T/A base pairs)+4(number G/C base pairs)(Suggs, S. V., T. Miyake, E. H. Kawashime, M. J. Johnson, K. Itakura,and R. B. Wallace [1981] ICN-UCLA Symp. Dev. Biol. Using Purified Genes,D. D. Brown [ed.], Academic Press, New York, 23:683-693).

[0070] Washes were typically carried out as follows:

[0071] (1) Twice at room temperature for 15 minutes 1X SSPE, 0.1% SDS(low stringency wash).

[0072] (2) Once at the hybridization temperature for 15 minutes in 1XSSPE, 0.1% SDS (moderate stringency wash).

[0073] The DNA sequences of the subject invention can also be used asprimers for PCR amplification. Polymerase Chain Reaction (PCR) is arepetitive, enzymatic, primed synthesis of a nucleic acid sequence. Thisprocedure is well known and commonly used by those skilled in this art(see Mullis, U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; Saiki,Randall K., Stephen Scharf, Fred Faloona, Kary B. Mullis, Glenn T. Horn,Henry A. Erlich, Norman Arnheim [1985] “Enzymatic Amplification ofβ-Globin Genomic Sequences and Restriction Site Analysis for Diagnosisof Sickle Cell Anemia,” Science 230:1350-1354). PCR is based on theenzymatic amplification of a DNA fragment of interest that is flanked bytwo oligonucleotide primers that hybridize to opposite strands of thetarget sequence. The primers are oriented with the 3 ends pointingtowards each other. Repeated cycles of heat denaturation of thetemplate, annealing of the primers to their complementary sequences, andextension of the annealed primers with a DNA polymerase result in theamplification of the segment defined by the 5 ends of the PCR primers.Since the extension product of each primer can serve as a template forthe other primer, each cycle essentially doubles the amount of DNAfragment produced in the previous cycle. This results in the exponentialaccumulation of the specific target fragment, up to several million-foldin a few hours. By using a thermostable DNA polymerase such as Taqpolymerase, which is isolated from the thermophilic bacterium Thermusaquaticus, the amplification process can be completely automated.

[0074] Certain toxins of the subject invention have been specificallyexemplified herein. Since these toxins are merely exemplary of thetoxins of the subject invention, it should be readily apparent that thesubject invention comprises variant or equivalent toxins (and nucleotidesequences coding for equivalent toxins) having the same or similarpesticidal activity of the exemplified toxin. Equivalent toxins willhave high amino acid homology with an exemplified toxin. This amino acididentity will typically be greater than 60%, preferably be greater than75%, more preferably greater than 80%, more preferably greater than 90%,and can be greater than 95%. These identities are as determined usingstandard alignment techniques. The amino acid homology will be highestin critical regions of the toxin which account for biological activityor are involved in the determination of three-dimensional configurationwhich ultimately is responsible for the biological activity. In thisregard, certain amino acid substitutions are acceptable and can beexpected if these substitutions are in regions which are not critical toactivity or are conservative amino acid substitutions which do notaffect the three-dimensional configuration of the molecule. For example,amino acids may be placed in the following classes: non-polar, unchargedpolar, basic, and acidic. Conservative substitutions whereby an aminoacid of one class is replaced with another amino acid of the same typefall within the scope of the subject invention so long as thesubstitution does not materially alter the biological activity of thecompound. Table 1 provides a listing of examples of amino acidsbelonging to each class. TABLE 1 Class of Amino Acid Examples of AminoAcids Nonpolar Ala, Val, Leu, Ile, Pro, Met, Phe, Trp Uncharged PolarGly, Ser, Thr, Cys, Tyr, Asn, Gln Acidic Asp, Glu Basic Lys, Arg, His

[0075] In some instances, non-conservative substitutions can also bemade. The critical factor is that these substitutions must notsignificantly detract from the biological activity of the toxin.

[0076] As used herein, reference to “isolated” polynucleotides and/or“purified” toxins refers to these molecules when they are not associatedwith the other molecules with which they would be found in nature. Thus,reference to “isolated and purified” signifies the involvement of the“hand of man” as described herein.

[0077] Recombinant hosts. The toxin-encoding genes of the subjectinvention can be introduced into a wide variety of microbial or planthosts. Expression of the toxin gene results, directly or indirectly, inthe intracellular production and maintenance of the pesticide. Withsuitable microbial hosts, e.g., Pseudomonas, the microbes can be appliedto the situs of the pest, where they will proliferate and be ingested.The result is a control of the pest. Alternatively, the microbe hostingthe toxin gene can be treated under conditions that prolong the activityof the toxin and stabilize the cell. The treated cell, which retains thetoxic activity, then can be applied to the environment of the targetpest.

[0078] Where the B.t. toxin gene is introduced via a suitable vectorinto a microbial host, and said host is applied to the environment in aliving state, it is essential that certain host microbes be used.Microorganism hosts are selected which are known to occupy the“phytosphere” (phylloplane, phyllosphere, rhizosphere, and/orrhizoplane) of one or more crops of interest.

[0079] These microorganisms are selected so as to be capable ofsuccessfully competing in the particular environment (crop and otherinsect habitats) with the wild-type microorganisms, provide for stablemaintenance and expression of the gene expressing the polypeptidepesticide, and, desirably, provide for improved protection of thepesticide from environmental degradation and inactivation.

[0080] A large number of microorganisms are known to inhabit thephylloplane (the surface of the plant leaves) and/or the rhizosphere(the soil surrounding plant roots) of a wide variety of important crops.These microorganisms include bacteria, algae, and fungi. Of particularinterest are microorganisms, such as bacteria, e.g., genera Pseudomonas,Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium,Rhodopseudomonas, Methylophilus, Agrobacterium, Acetobacter,Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes;fungi, particularly yeast, e.g., genera Saccharomyces, Cryptococcus,Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Ofparticular interest are such phytosphere bacterial species asPseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens,Acetobacter xylinum, Agrobacterium tumefaciens, Rhodopseudomonasspheroides, Xanthomonas campestris, Rhizobium meliloti, Alcaligeneseutrophus, and Azotobacter vinlandii; and phytosphere yeast species suchas Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca,Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei,S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus,Kluyveromyces veronae, and Aureobasidium pullulans. Of particularinterest are the pigmented microorganisms.

[0081] A significant number of methods are available for introducing aB.t. gene encoding a toxin into a microorganism host under conditionswhich allow for stable maintenance and expression of the gene. Thesemethods are well known to those skilled in the art and are described,for example, in U.S. Pat. No. 5,135,867, which is incorporated herein byreference.

[0082] Control of coleopterans, including corn rootworm, as well asnematodes, using the isolates, toxins, and genes of the subjectinvention can be accomplished by a variety of methods known to thoseskilled in the art. These methods include, for example, the applicationof B.t. isolates to the pests (or their location), the application ofrecombinant microbes to the pests (or their locations), and thetransformation of plants with genes which encode the pesticidal toxinsof the subject invention. Recombinant microbes may be, for example, aB.t., E. coli, or Pseudomonas. Transformations can be made by thoseskilled in the art using standard techniques. Materials necessary forthese transformations are disclosed herein or are otherwise readilyavailable to the skilled artisan.

[0083] Synthetic genes which are functionally equivalent to the genesexemplified herein can also be used to transform hosts. Methods for theproduction of synthetic genes can be found in, for example, U.S. Pat.No. 5,380,831.

[0084] Treatment of cells. As mentioned above, B.t. or recombinant cellsexpressing a B.t. toxin can be treated to prolong the toxin activity andstabilize the cell. The pesticide microcapsule that is formed comprisesthe B.t. toxin within a cellular structure that has been stabilized andwill protect the toxin when the microcapsule is applied to theenvironment of the target pest. Suitable host cells may include eitherprokaryotes or eukaryotes, normally being limited to those cells whichdo not produce substances toxic to higher organisms, such as mammals.However, organisms which produce substances toxic to higher organismscould be used, where the toxic substances are unstable or the level ofapplication sufficiently low as to avoid any possibility of toxicity toa mammalian host. As hosts, of particular interest will be theprokaryotes and the lower eukaryotes, such as fungi.

[0085] The cell will usually be intact and be substantially in theproliferative form when treated, rather than in a spore form, althoughin some instances spores may be employed.

[0086] Treatment of the microbial cell, e.g., a microbe containing theB.t. toxin gene, can be by chemical or physical means, or by acombination of chemical and/or physical means, so long as the techniquedoes not deleteriously affect the properties of the toxin, nor diminishthe cellular capability of protecting the toxin. Examples of chemicalreagents are halogenating agents, particularly halogens of atomic no.17-80. More particularly, iodine can be used under mild conditions andfor sufficient time to achieve the desired results. Other suitabletechniques include treatment with aldehydes, such as glutaraldehyde;anti-infectives, such as zephiran chloride and cetylpyridinium chloride;alcohols, such as isopropyl and ethanol; various histologic fixatives,such as Lugol iodine, Bouin's fixative, various acids and Helly'sfixative (See: Humason, Gretchen L., Animal Tissue Techniques, W. H.Freeman and Company, 1967); or a combination of physical (heat) andchemical agents that preserve and prolong the activity of the toxinproduced in the cell when the cell is administered to the hostenvironment. Examples of physical means are short wavelength radiationsuch as gamma-radiation and X-radiation, freezing, UV irradiation,lyophilization, and the like. Methods for treatment of microbial cellsare disclosed in U.S. Pat. Nos. 4,695,455 and 4,695,462, which areincorporated herein by reference.

[0087] The cells generally will have enhanced structural stability whichwill enhance resistance to environmental conditions. Where the pesticideis in a proform, the method of cell treatment should be selected so asnot to inhibit processing of the proform to the mature form of thepesticide by the target pest pathogen. For example, formaldehyde willcrosslink proteins and could inhibit processing of the proform of apolypeptide pesticide. The method of treatment should retain at least asubstantial portion of the bio-availability or bioactivity of the toxin.

[0088] Characteristics of particular interest in selecting a host cellfor purposes of production include ease of introducing the B.t. geneinto the host, availability of expression systems, efficiency ofexpression, stability of the pesticide in the host, and the presence ofauxiliary genetic capabilities. Characteristics of interest for use as apesticide microcapsule include protective qualities for the pesticide,such as thick cell walls, pigmentation, and intracellular packaging orformation of inclusion bodies; survival in aqueous environments; lack ofmammalian toxicity; attractiveness to pests for ingestion; ease ofkilling and fixing without damage to the toxin; and the like. Otherconsiderations include ease of formulation and handling, economics,storage stability, and the like.

[0089] Growth of cells. The cellular host containing the B.t.insecticidal gene may be grown in any convenient nutrient medium, wherethe DNA construct provides a selective advantage, providing for aselective medium so that substantially all or all of the cells retainthe B.t. gene. These cells may then be harvested in accordance withconventional ways. Alternatively, the cells can be treated prior toharvesting.

[0090] The B.t. cells of the invention can be cultured using standardart media and fermentation techniques. Upon completion of thefermentation cycle the bacteria can be harvested by first separating theB.t. spores and crystals from the fermentation broth by means well knownin the art. The recovered B.t. spores and crystals can be formulatedinto a wettable powder, liquid concentrate, granules or otherformulations by the addition of surfactants, dispersants, inertcarriers, and other components to facilitate handling and applicationfor particular target pests. These formulations and applicationprocedures are all well known in the art.

[0091] Methods and formulations for control of pests. Control ofcoleopterans using the toxins and genes of the subject invention can beaccomplished by a variety of methods known to those skilled in the art.These methods include, for example, the application of B.t. isolates tothe pests (or their location), the application of recombinant microbesto the pests (or their locations), and the transformation of plants withgenes which encode the pesticidal toxins of the subject invention.Recombinant microbes may be, for example, a B.t., E. coli, orPseudomonas. Transformations can be made by those skilled in the artusing standard techniques. Materials necessary for these transformationsare disclosed herein or are otherwise readily available to the skilledartisan.

[0092] Formulated bait granules containing an attractant and spores andcrystals of the B.t. isolates, or recombinant microbes comprising thegenes obtainable from the B.t. isolates disclosed herein, can be appliedto the soil. Formulated product can also be applied as a seed-coating orroot treatment or total plant treatment at later stages of the cropcycle. Plant and soil treatments of B.t. cells may be employed aswettable powders, granules or dusts, by mixing with various inertmaterials, such as inorganic minerals (phyllosilicates, carbonates,sulfates, phosphates, and the like) or botanical materials (powderedcorncobs, rice hulls, walnut shells, and the like). The formulations mayinclude spreader-sticker adjuvants, stabilizing agents, other pesticidaladditives, or surfactants. Liquid formulations may be aqueous-based ornon-aqueous and employed as foams, gels, suspensions, emulsifiableconcentrates, or the like. The ingredients may include Theologicalagents, surfactants, emulsifiers, dispersants, or polymers.

[0093] As would be appreciated by a person skilled in the art, thepesticidal concentration will vary widely depending upon the nature ofthe particular formulation, particularly whether it is a concentrate orto be used directly. The pesticide will be present in at least 1% byweight and may be 100% by weight. The dry formulations will have fromabout 1-95% by weight of the pesticide while the liquid formulationswill generally be from about 1-60% by weight of the solids in the liquidphase. The formulations will generally have from about 10² to about 10⁴cells/mg. These formulations will be administered at about 50 mg (liquidor dry) to 1 kg or more per hectare.

[0094] The formulations can be applied to the environment of the pest,e.g., soil and foliage, by spraying, dusting, sprinkling, or the like.

[0095] All of the U.S. Pat. Nos. referred to herein are herebyincorporated by reference.

[0096] Following are examples which illustrate procedures for practicingthe invention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLES1 Culturing B.t. Isolates

[0097] A subculture of the B.t. isolate can be used to inoculate thefollowing medium, a peptone, glucose, salts medium. Bacto Peptone 7.5g/l Glucose 1.0 g/l KH₂PO₄ 3.4 g/l K₂HPO₄ 4.35 g/l Salts Solution 5.0ml/l CaCl₂ Solution 5.0 ml/l Salts Solution (100 ml) MgSO₄ 7H₂O 2.46 gMnSO₄ H₂O 0.04 g ZnSO₄ 7H₂O 0.28 g FeSO₄ 7H₂O 0.40 g CaCl₂ Solution (100ml) CaCl₂ 2H₂O 3.66 g pH 7.2

[0098] The salts solution and CaCl₂ solution are filter-sterilized andadded to the autoclaved and cooked broth at the time of inoculation.Flasks are incubated at 30° C. on a rotary shaker at 200 rpm for 64 hr.

[0099] The above procedure can be readily scaled up to large fermentorsby procedures well known in the art.

[0100] The B.t. spores and crystals, obtained in the above fermentation,can be isolated by procedures well known in the art. A frequently-usedprocedure is to subject the harvested fermentation broth to separationtechniques, e.g., centrifugation.

EXAMPLE 2 Expression and Purification of Recombinant Cry6A Toxin fromStrain PS86A1

[0101] Starter culture was prepared consisting of 50 ml of autoclaved LBmedium contained in a 250 ml baffled culture flask, inoculated with 50μl of PS86A1. The flask was closed and incubated at 30° C. on a rotaryshaker at 225 rpm for 4-6 hours. Five milliliters of the starter wasthen used to inoculate 300 ml of autoclaved growth medium in a 2-literbaffled culture flask with foam plugs. The growth medium is designatedNYS-CAA and consists of: Nutrient broth (Difco) 3.75 g Tryptone 3.75 gCasamino acids 6.00 g Yeast extract 1.50 g B.t. salts 30 ml The B.t.salts stock solution consists of: CaCl₂ 2H₂O 10.30 g MgCl₂ 6H₂O 20.35 gMnCl₂ 4H₂O 1.00 g FeSO₄ 7H₂O 0.02 g ZnSO₄ 7H₂O 0.02 g (NH₄)₂SO₄ 0.02 gHCl (7N) 1.00 ml

[0102] The inoculated culture was grown on a large rotary shaker at 30°C. for up to 65 hours or more (until lysis is substantially complete).The particulates were harvested by centrifugation at 4° C. and 8,000 rpmin a Sorvall GS-3 rotor. The resulting pellet was washed three timeswith approximately 5 volumes of distilled water by resuspending thepelleted material and centrifuging as described. Toxin crystals werepurified using centrifugation (100 minutes at 6,500 rpm in a Sorval HS-4rotor at 4° C.) over sodium bromide step gradients consisting of 15 ml50%, 7 ml of 45%, and 7 ml of 40% NaBr. Crystals were removed from thegradients, diluted approximately 50% with distilled water, andconcentrated by centrifugation at 14,000 rpm at 4° C. in a Sorvall SS-34rotor. The crystal protein pellet was suspended in a minimal amount ofdistilled water, quick frozen in a dry ice/isopropanol bath, and storedat 80° C. SDS-PAGE analysis of the products of this process reveals adominant 54 kDa Coomassie staining band. Analysis by mass spectroscopy(matrix-assisted laser desorbtion-time of flight, MALDI-TOF) of theprotein detects a dominant peak at 54,080 daltons. The molecular weightcalculated from the amino acid sequence of the intact 86A1 toxin is54,080 daltons.

EXAMPLE 3 Proteolytic Digestion of Cry6A Toxin

[0103] The crystal proteins obtained as above were proteolyticallydigested using bovine trypsin. The digestion mixture contained 133 mMTris base, 1 M urea, 5 mg of 86A1 toxin protein, 50 μg of trypsin (e.g.,Sigma Type XIII or Boerhinger-Mannheim sequencing grade), in 2.0 mlfinal volume. The above mixture minus trypsin was incubated at 37° C.for 15 minutes. Trypsin, as a 1 mg/ml solution in 10 mM sodium acetate,pH 4.5, was then added and allowed to incubate an additional two hoursat 37° C. At the conclusion of the incubation, the reaction mixture wascentrifuged for 15 minutes in an Eppendorf centrifuge at 4° C. Thesupernatant was removed and placed in a 2-ml Centricon 30 (Amicon) andwashed three times with 2 ml of distilled water. The washed sample wasstored at 4° C. or was quick frozen in a dry ice/isopropanol bath andstored at 80° C.

[0104] SDS-PAGE analysis of the washed digestion mixture reveals adominant Coomassie staining band at 45,000 daltons, with minor bandsdetectable at 46,000 and daltons and approximately 34,000 daltons.MALDI-TOF reveals a single band at 46,500 daltons. Amino terminalsequence analysis using automated (ABI) Edman degradation of theSDS-PAGE band blotted to a PVDF membrane, reveals an 11-amino acidsequence which corresponds to the known sequence of the full-length 86A1toxin (SEQ ID NO. 2) starting at amino acid residue number 11. Automatedcarboxyl terminal sequencing (HP) of the major SDS-PAGE band blotted toa Zitex membrane revealed a sequence which corresponds to the knownsequence of 86A1 toxin beginning at amino acid residue 441 and ending atamino acid residue 443 of SEQ ID NO. 2. The calculated mass of thesequenced fragment (residue 12-443) is 48,725 daltons.

[0105] This resulting truncated toxin is referred to herein as R443, thetruncated 86A1 toxin, or the truncated Cry6A toxin. The sequence of thistoxin was determined to be that of SEQ ID NO. 6. See also SEQ ID NO. 5.

[0106] In addition to the above preferred method, a similar result canbe obtained in the absence of 1 M urea. If 140 mM β-mercaptoethanol isadded, either in the presence or absence of 1 M urea, the yield ofproduct is much reduced. This can be overcome, to some extent, in theabsence of urea by increasing the amount of trypsin 10-fold. It islikely that any buffer having a pH between pH 9-11 would performsimilarly.

EXAMPLE 4 Western Corn Rootworm Bioassay

[0107] The truncated protein preparations obtained as described above inExample 3 were bioassayed against western corn rootworm and were foundto have significant toxin activity.

[0108] As shown in Table 2, the levels of activity obtained by using thetruncated 86A1 protein unexpectedly exceed the control levels obtainedby using the full length 86A1 protein. TABLE 2 MORTALITY Dosage Test 1Test 2 Treatment (μg/cm²) dead/total dead/total Average % Truncated 86A1475 10/10 6/7 93 activated protein 237 7/10 10/10 85 118 6/10 9/11 71 590/10 5/11 23 29 6/16 2/16 25 Full length 86A1 481 1/16 3/13 14 protein240 3/13 4/12 28 120 4/24 1/12 12 60 1/13 0/10 3 30 6/24 2/16 18 Buffercontrol 1 X 0/8 0/9 0 0.5 X 1/8 0/11 6 0.25 X 3/8 0/11 18 0.12 X 0/110/10 0 0.06 X 6/23 0/12 13

[0109] The LC50 (μg/cm²) was determined for the original length (58kDa)86A1 protein and for the truncated form (45 kDa) of the 86A1 protein.The results are reported in Table 3. TABLE 3 Protein Process Size ofprotein LC50 μg/cm² 86A1 Purified 58 kDa not lethal solubilized 86A1purified 45 kDa 77 solubilized trypsin digested

EXAMPLE 5 Construction of Fusion Proteins and Genes

[0110] A fusion protein consisting of Cry6B and Cry6A having activityagainst western corn rootworm can be constructed. It should be notedthat the Cry6B/69D1 protein was not previously known to be useful forcontrolling corn rootworm. The sequence of the fall length Cry6B toxinobtainable from PS69D1 corresponds to SEQ ID NO. 10. See also SEQ ID NO.9.

[0111] The fusion consists of a gene segment from Cry6B joined to onefrom Cry6A such that the open reading frame is maintained. Oneembodiment consists of joining amino acids 1 through 394 of Cry6B toamino acids 386 through 443 of Cry6A. It would be preferable to make acrossover between the two genes at a point where there is substantialhomology, because substantial homology infers similar three-dimensionalstructure and the possibility of fewer detrimental perturbations inthree-dimensional structure. FIG. 1 is an alignment of Cry6A to Cry6Bshowing regions of homology for crossovers. Other possible examples ofcrossover points for gene fusions are shown below in Table 2. The tableis not to be construed as limiting. TABLE 4 Cry6B amino acids Cry6Aamino acids 1-394 386-443 1-248 241-443 1-264 256-443 1-302 295-4431-395 387-443

[0112] Additionally, the constructs may be truncated on the aminoterminus so that the first approximately 10-25 amino acids may beabsent.

EXAMPLE 6 Insertion of Toxin Genes Into Plants

[0113] One aspect of the subject invention is the transformation ofplants with the subject genes encoding the insecticidal toxin. Thetransformed plants are resistant to attack by the target pest. Inpreferred embodiments, the truncated genes of the subject invention areoptimized for use in plants. SEQ ID NOS. 3 and 4 provide sequenceinformation for plant-optimized versions of the full-length cry6A geneand toxin. The truncated genes can also be optimized for use in plants.For example, the gene and toxin sequences of SEQ ID NOS. 7 and 8, whichcan also be referred to as R390, are optimized for use in plants.

[0114] Genes encoding pesticidal toxins, as disclosed herein, can beinserted into plant cells using a variety of techniques which are wellknown in the art. For example, a large number of cloning vectorscomprising a replication system in E. coli and a marker that permitsselection of the transformed cells are available for preparation for theinsertion of foreign genes into higher plants. The vectors comprise, forexample, pBR322, pUC series, M13mp series, pACYC184, etc. Accordingly,the sequence encoding the B.t. toxin can be inserted into the vector ata suitable restriction site. The resulting plasmid is used fortransformation into E. coli. The E. coli cells are cultivated in asuitable nutrient medium, then harvested and lysed. The plasmid isrecovered. Sequence analysis, restriction analysis, electrophoresis, andother biochemical-molecular biological methods are generally carried outas methods of analysis. After each manipulation, the DNA sequence usedcan be cleaved and joined to the next DNA sequence. Each plasmidsequence can be cloned in the same or other plasmids. Depending on themethod of inserting desired genes into the plant, other DNA sequencesmay be necessary. If, for example, the Ti or Ri plasmid is used for thetransformation of the plant cell, then at least the right border, butoften the right and the left border of the Ti or Ri plasmid T-DNA, hasto be joined as the flanking region of the genes to be inserted.

[0115] The use of T-DNA for the transformation of plant cells has beenintensively researched and sufficiently described in EP 120 516; Hoekema(1985) In: The Binary Plant Vector System, Offset-durkkerij Kanters B.V., Alblasserdam, Chapter 5; Fraley et al., Crit. Rev. Plant Sci. 4:1-46; and An et al. (1985) EMBO J. 4:277-287.

[0116] Once the inserted DNA has been integrated in the genome, it isrelatively stable there and, as a rule, does not come out again. Itnormally contains a selection marker that confers on the transformedplant cells resistance to a biocide or an antibiotic, such as kanamycin,G 418, bleomycin, hygromycin, or chloramphenicol, inter alia. Theindividually employed marker should accordingly permit the selection oftransformed cells rather than cells that do not contain the insertedDNA.

[0117] A large number of techniques are available for inserting DNA intoa plant host cell. Those techniques include transformation with T-DNAusing Agrobacterium tumefaciens or Agrobacterium rhizogenes astransformation agent, fusion, injection, biolistics (microparticlebombardment), or electroporation as well as other possible methods. IfAgrobacteria are used for the transformation, the DNA to be inserted hasto be cloned into special plasmids, namely either into an intermediatevector or into a binary vector. The intermediate vectors can beintegrated into the Ti or Ri plasmid by homologous recombination owingto sequences that are homologous to sequences in the T-DNA. The Ti or Riplasmid also comprises the vir region necessary for the transfer of theT-DNA. Intermediate vectors cannot replicate themselves in Agrobacteria.The intermediate vector can be transferred into Agrobacteriumtumefaciens by means of a helper plasmid (conjugation). Binary vectorscan replicate themselves both in E. coli and in Agrobacteria. Theycomprise a selection marker gene and a linker or polylinker which areframed by the right and left T-DNA border regions. They can betransformed directly into Agrobacteria (Holsters et al. [1978] Mol. Gen.Genet. 163:181-187). The Agrobacterium used as host cell is to comprisea plasmid carrying a vir region. The vir region is necessary for thetransfer of the T-DNA into the plant cell. Additional T-DNA may becontained. The bacterium so transformed is used for the transformationof plant cells. Plant explants can advantageously be cultivated withAgrobacterium tumefaciens or Agrobacterium rhizogenes for the transferof the DNA into the plant cell. Whole plants can then be regeneratedfrom the infected plant material (for example, pieces of leaf, segmentsof stalk, roots, but also protoplasts or suspension-cultivated cells) ina suitable medium, which may contain antibiotics or biocides forselection. The plants so obtained can then be tested for the presence ofthe inserted DNA. No special demands are made of the plasmids in thecase of injection and electroporation. It is possible to use ordinaryplasmids, such as, for example, pUC derivatives.

[0118] The transformed cells grow inside the plants in the usual manner.They can form germ cells and transmit the transformed trait(s) toprogeny plants. Such plants can be grown in the normal manner andcrossed with plants that have the same transformed hereditary factors orother hereditary factors. The resulting hybrid individuals have thecorresponding phenotypic properties.

[0119] In a preferred embodiment of the subject invention, plants willbe transformed with genes wherein the codon usage has been optimized forplants. See, for example, U.S. Pat. No. 5,380,831. Also, advantageously,plants encoding a truncated toxin will be used. The truncated toxintypically will encode about 55% to about 80% of the full length toxin.Methods for creating synthetic B.t. genes for use in plants are known inthe art.

[0120] It should be understood that the examples and embodimentsdescribed herein are for illustrative purposes only and that variousmodifications or changes in light thereof will be suggested to personsskilled in the art and are to be included within the spirit and purviewof this application and the scope of the appended claims.

1 10 1 1425 DNA Bacillus thuringiensis 1 atgattattg atagtaaaacgactttacct agacattcac ttattcatac aattaaatta 60 aattctaata agaaatatggtcctggtgat atgactaatg gaaatcaatt tattatttca 120 aaacaagaat gggctacgattggagcatat attcagactg gattaggttt accagtaaat 180 gaacaacaat taagaacacatgttaattta agtcaggata tatcaatacc tagtgatttt 240 tctcaattat atgatgtttattgttctgat aaaacttcag cagaatggtg gaataaaaat 300 ttatatcctt taattattaaatctgctaat gatattgctt catatggttt taaagttgct 360 ggtgatcctt ctattaagaaagatggatat tttaaaaaat tgcaagatga attagataat 420 attgttgata ataattccgatgatgatgca atagctaaag ctattaaaga ttttaaagcg 480 cgatgtggta ttttaattaaagaagctaaa caatatgaag aagctgcaaa aaatattgta 540 acatctttag atcaatttttacatggtgat cagaaaaaat tagaaggtgt tatcaatatt 600 caaaaacgtt taaaagaagttcaaacagct cttaatcaag cccatgggga aagtagtcca 660 gctcataaag agttattagaaaaagtaaaa aatttaaaaa caacattaga aaggactatt 720 aaagctgaac aagatttagagaaaaaagta gaatatagtt ttctattagg accattgtta 780 ggatttgttg tttatgaaattcttgaaaat actgctgttc agcatataaa aaatcaaatt 840 gatgagataa agaaacaattagattctgct cagcatgatt tggatagaga tgttaaaatt 900 ataggaatgt taaatagtattaatacagat attgataatt tatatagtca aggacaagaa 960 gcaattaaag ttttccaaaagttacaaggt atttgggcta ctattggagc tcaaatagaa 1020 aatcttagaa caacgtcgttacaagaagtt caagattctg atgatgctga tgagatacaa 1080 attgaacttg aggacgcttctgatgcttgg ttagttgtgg ctcaagaagc tcgtgatttt 1140 acactaaatg cttattcaactaatagtaga caaaatttac cgattaatgt tatatcagat 1200 tcatgtaatt gttcaacaacaaatatgaca tcaaatcaat acagtaatcc aacaacaaat 1260 atgacatcaa atcaatatatgatttcacat gaatatacaa gtttaccaaa taattttatg 1320 ttatcaagaa atagtaatttagaatataaa tgtcctgaaa ataattttat gatatattgg 1380 tataataatt cggattggtataataattcg gattggtata ataat 1425 2 475 PRT Bacillus thuringiensis 2 MetIle Ile Asp Ser Lys Thr Thr Leu Pro Arg His Ser Leu Ile His 1 5 10 15Thr Ile Lys Leu Asn Ser Asn Lys Lys Tyr Gly Pro Gly Asp Met Thr 20 25 30Asn Gly Asn Gln Phe Ile Ile Ser Lys Gln Glu Trp Ala Thr Ile Gly 35 40 45Ala Tyr Ile Gln Thr Gly Leu Gly Leu Pro Val Asn Glu Gln Gln Leu 50 55 60Arg Thr His Val Asn Leu Ser Gln Asp Ile Ser Ile Pro Ser Asp Phe 65 70 7580 Ser Gln Leu Tyr Asp Val Tyr Cys Ser Asp Lys Thr Ser Ala Glu Trp 85 9095 Trp Asn Lys Asn Leu Tyr Pro Leu Ile Ile Lys Ser Ala Asn Asp Ile 100105 110 Ala Ser Tyr Gly Phe Lys Val Ala Gly Asp Pro Ser Ile Lys Lys Asp115 120 125 Gly Tyr Phe Lys Lys Leu Gln Asp Glu Leu Asp Asn Ile Val AspAsn 130 135 140 Asn Ser Asp Asp Asp Ala Ile Ala Lys Ala Ile Lys Asp PheLys Ala 145 150 155 160 Arg Cys Gly Ile Leu Ile Lys Glu Ala Lys Gln TyrGlu Glu Ala Ala 165 170 175 Lys Asn Ile Val Thr Ser Leu Asp Gln Phe LeuHis Gly Asp Gln Lys 180 185 190 Lys Leu Glu Gly Val Ile Asn Ile Gln LysArg Leu Lys Glu Val Gln 195 200 205 Thr Ala Leu Asn Gln Ala His Gly GluSer Ser Pro Ala His Lys Glu 210 215 220 Leu Leu Glu Lys Val Lys Asn LeuLys Thr Thr Leu Glu Arg Thr Ile 225 230 235 240 Lys Ala Glu Gln Asp LeuGlu Lys Lys Val Glu Tyr Ser Phe Leu Leu 245 250 255 Gly Pro Leu Leu GlyPhe Val Val Tyr Glu Ile Leu Glu Asn Thr Ala 260 265 270 Val Gln His IleLys Asn Gln Ile Asp Glu Ile Lys Lys Gln Leu Asp 275 280 285 Ser Ala GlnHis Asp Leu Asp Arg Asp Val Lys Ile Ile Gly Met Leu 290 295 300 Asn SerIle Asn Thr Asp Ile Asp Asn Leu Tyr Ser Gln Gly Gln Glu 305 310 315 320Ala Ile Lys Val Phe Gln Lys Leu Gln Gly Ile Trp Ala Thr Ile Gly 325 330335 Ala Gln Ile Glu Asn Leu Arg Thr Thr Ser Leu Gln Glu Val Gln Asp 340345 350 Ser Asp Asp Ala Asp Glu Ile Gln Ile Glu Leu Glu Asp Ala Ser Asp355 360 365 Ala Trp Leu Val Val Ala Gln Glu Ala Arg Asp Phe Thr Leu AsnAla 370 375 380 Tyr Ser Thr Asn Ser Arg Gln Asn Leu Pro Ile Asn Val IleSer Asp 385 390 395 400 Ser Cys Asn Cys Ser Thr Thr Asn Met Thr Ser AsnGln Tyr Ser Asn 405 410 415 Pro Thr Thr Asn Met Thr Ser Asn Gln Tyr MetIle Ser His Glu Tyr 420 425 430 Thr Ser Leu Pro Asn Asn Phe Met Leu SerArg Asn Ser Asn Leu Glu 435 440 445 Tyr Lys Cys Pro Glu Asn Asn Phe MetIle Tyr Trp Tyr Asn Asn Ser 450 455 460 Asp Trp Tyr Asn Asn Ser Asp TrpTyr Asn Asn 465 470 475 3 1428 DNA Bacillus thuringiensis 3 atggtcattgacagcaagac gactctacca cggcactcac tgattcacac aatcaagctg 60 aactctaacaagaagtatgg tcctggcgat atgactaacg ggaaccagtt catcatatcc 120 aagcaagaatgggccacgat tggcgcatac attcagactg gactcggctt accagtgaat 180 gagcaacagctgagaaccca cgttaacctt agtcaagaca tcagcatacc atctgacttt 240 tctcaactctacgatgtgta ttgttctgac aagactagtg cagaatggtg gaacaagaat 300 ctctatcctttgatcatcaa gtctgccaat gacattgctt catatggttt caaagttgct 360 ggtgatccttcgatcaagaa agatggttac ttcaagaagc ttcaagatga actcgacaac 420 attgttgacaacaactccga cgacgatgcg atagccaaag ccatcaagga cttcaaagca 480 agatgtggcattctcatcaa ggaagccaag cagtatgaag aagctgccaa gaacattgta 540 acatcattggatcagtttct ccatggagac cagaagaagc tcgagggtgt catcaacatt 600 cagaaacgtctgaaagaggt tcaaacagct ctgaatcaag cccatgggga atccagtcca 660 gctcacaaagagcttcttga gaaagtgaag aacttgaaga ccacacttga gaggaccatc 720 aaagctgaacaagacttgga gaagaaagta gagtacagct ttctacttgg acccttgtta 780 ggctttgttgtctacgagat tcttgagaac actgctgttc aacacatcaa gaatcaaatc 840 gatgagatcaagaaacagtt ggattctgcg caacatgact tggatcgcga tgtgaagatc 900 attggaatgctcaacagcat caacactgac attgacaact tgtatagtca aggacaagaa 960 gcaatcaaagtctttcagaa gctacaaggg atatgggcca ctattggagc tcagatagag 1020 aatcttcgcaccacgtccct tcaagaagtc caagactctg atgatgctga tgagatacag 1080 attgaacttgaggacgcatc tgatgcatgg ttagttgtgg ctcaagaagc tcgtgacttc 1140 acactgaatgcctactcaac caacagtcga cagaatctcc cgatcaatgt gatctcagat 1200 tcatgcaattgctccaccac caacatgaca tccaatcagt acagcaatcc aacaaccaac 1260 atgactagcaatcagtacat gattagccat gagtatacca gcttgcccaa caacttcatg 1320 ttgtcaaggaattcgaacct ggagtacaag tgccctgaga acaacttcat gatctactgg 1380 tacaacaactccgattggta caacaattcg gattggtaca acaattaa 1428 4 475 PRT Bacillusthuringiensis 4 Met Val Ile Asp Ser Lys Thr Thr Leu Pro Arg His Ser LeuIle His 1 5 10 15 Thr Ile Lys Leu Asn Ser Asn Lys Lys Tyr Gly Pro GlyAsp Met Thr 20 25 30 Asn Gly Asn Gln Phe Ile Ile Ser Lys Gln Glu Trp AlaThr Ile Gly 35 40 45 Ala Tyr Ile Gln Thr Gly Leu Gly Leu Pro Val Asn GluGln Gln Leu 50 55 60 Arg Thr His Val Asn Leu Ser Gln Asp Ile Ser Ile ProSer Asp Phe 65 70 75 80 Ser Gln Leu Tyr Asp Val Tyr Cys Ser Asp Lys ThrSer Ala Glu Trp 85 90 95 Trp Asn Lys Asn Leu Tyr Pro Leu Ile Ile Lys SerAla Asn Asp Ile 100 105 110 Ala Ser Tyr Gly Phe Lys Val Ala Gly Asp ProSer Ile Lys Lys Asp 115 120 125 Gly Tyr Phe Lys Lys Leu Gln Asp Glu LeuAsp Asn Ile Val Asp Asn 130 135 140 Asn Ser Asp Asp Asp Ala Ile Ala LysAla Ile Lys Asp Phe Lys Ala 145 150 155 160 Arg Cys Gly Ile Leu Ile LysGlu Ala Lys Gln Tyr Glu Glu Ala Ala 165 170 175 Lys Asn Ile Val Thr SerLeu Asp Gln Phe Leu His Gly Asp Gln Lys 180 185 190 Lys Leu Glu Gly ValIle Asn Ile Gln Lys Arg Leu Lys Glu Val Gln 195 200 205 Thr Ala Leu AsnGln Ala His Gly Glu Ser Ser Pro Ala His Lys Glu 210 215 220 Leu Leu GluLys Val Lys Asn Leu Lys Thr Thr Leu Glu Arg Thr Ile 225 230 235 240 LysAla Glu Gln Asp Leu Glu Lys Lys Val Glu Tyr Ser Phe Leu Leu 245 250 255Gly Pro Leu Leu Gly Phe Val Val Tyr Glu Ile Leu Glu Asn Thr Ala 260 265270 Val Gln His Ile Lys Asn Gln Ile Asp Glu Ile Lys Lys Gln Leu Asp 275280 285 Ser Ala Gln His Asp Leu Asp Arg Asp Val Lys Ile Ile Gly Met Leu290 295 300 Asn Ser Ile Asn Thr Asp Ile Asp Asn Leu Tyr Ser Gln Gly GlnGlu 305 310 315 320 Ala Ile Lys Val Phe Gln Lys Leu Gln Gly Ile Trp AlaThr Ile Gly 325 330 335 Ala Gln Ile Glu Asn Leu Arg Thr Thr Ser Leu GlnGlu Val Gln Asp 340 345 350 Ser Asp Asp Ala Asp Glu Ile Gln Ile Glu LeuGlu Asp Ala Ser Asp 355 360 365 Ala Trp Leu Val Val Ala Gln Glu Ala ArgAsp Phe Thr Leu Asn Ala 370 375 380 Tyr Ser Thr Asn Ser Arg Gln Asn LeuPro Ile Asn Val Ile Ser Asp 385 390 395 400 Ser Cys Asn Cys Ser Thr ThrAsn Met Thr Ser Asn Gln Tyr Ser Asn 405 410 415 Pro Thr Thr Asn Met ThrSer Asn Gln Tyr Met Ile Ser His Glu Tyr 420 425 430 Thr Ser Leu Pro AsnAsn Phe Met Leu Ser Arg Asn Ser Asn Leu Glu 435 440 445 Tyr Lys Cys ProGlu Asn Asn Phe Met Ile Tyr Trp Tyr Asn Asn Ser 450 455 460 Asp Trp TyrAsn Asn Ser Asp Trp Tyr Asn Asn 465 470 475 5 1299 DNA Bacillusthuringiensis 5 atgggtctga ttcacacaat caagctgaac tctaacaaga agtatggtcctggcgatatg 60 actaacggga accagttcat catatccaag caagaatggg ccacgattggcgcatacatt 120 cagactggac tcggcttacc agtgaatgag caacagctga gaacccacgttaaccttagt 180 caagacatca gcataccatc tgacttttct caactctacg atgtgtattgttctgacaag 240 actagtgcag aatggtggaa caagaatctc tatcctttga tcatcaagtctgccaatgac 300 attgcttcat atggtttcaa agttgctggt gatccttcga tcaagaaagatggttacttc 360 aagaagcttc aagatgaact cgacaacatt gttgacaaca actccgacgacgatgcgata 420 gccaaagcca tcaaggactt caaagcaaga tgtggcattc tcatcaaggaagccaagcag 480 tatgaagaag ctgccaagaa cattgtaaca tcattggatc agtttctccatggagaccag 540 aagaagctcg agggtgtcat caacattcag aaacgtctga aagaggttcaaacagctctg 600 aatcaagccc atggggaatc cagtccagct cacaaagagc ttcttgagaaagtgaagaac 660 ttgaagacca cacttgagag gaccatcaaa gctgaacaag acttggagaagaaagtagag 720 tacagctttc tacttggacc cttgttaggc tttgttgtct acgagattcttgagaacact 780 gctgttcaac acatcaagaa tcaaatcgat gagatcaaga aacagttggattctgcgcaa 840 catgacttgg atcgcgatgt gaagatcatt ggaatgctca acagcatcaacactgacatt 900 gacaacttgt atagtcaagg acaagaagca atcaaagtct ttcagaagctacaagggata 960 tgggccacta ttggagctca gatagagaat cttcgcacca cgtcccttcaagaagtccaa 1020 gactctgatg atgctgatga gatacagatt gaacttgagg acgcatctgatgcatggtta 1080 gttgtggctc aagaagctcg tgacttcaca ctgaatgcct actcaaccaacagtcgacag 1140 aatctcccga tcaatgtgat ctcagattca tgcaattgct ccaccaccaacatgacatcc 1200 aatcagtaca gcaatccaac aaccaacatg actagcaatc agtacatgattagccatgag 1260 tataccagct tgcccaacaa cttcatgttg tcaaggtag 1299 6 432PRT Bacillus thuringiensis 6 Met Gly Leu Ile His Thr Ile Lys Leu Asn SerAsn Lys Lys Tyr Gly 1 5 10 15 Pro Gly Asp Met Thr Asn Gly Asn Gln PheIle Ile Ser Lys Gln Glu 20 25 30 Trp Ala Thr Ile Gly Ala Tyr Ile Gln ThrGly Leu Gly Leu Pro Val 35 40 45 Asn Glu Gln Gln Leu Arg Thr His Val AsnLeu Ser Gln Asp Ile Ser 50 55 60 Ile Pro Ser Asp Phe Ser Gln Leu Tyr AspVal Tyr Cys Ser Asp Lys 65 70 75 80 Thr Ser Ala Glu Trp Trp Asn Lys AsnLeu Tyr Pro Leu Ile Ile Lys 85 90 95 Ser Ala Asn Asp Ile Ala Ser Tyr GlyPhe Lys Val Ala Gly Asp Pro 100 105 110 Ser Ile Lys Lys Asp Gly Tyr PheLys Lys Leu Gln Asp Glu Leu Asp 115 120 125 Asn Ile Val Asp Asn Asn SerAsp Asp Asp Ala Ile Ala Lys Ala Ile 130 135 140 Lys Asp Phe Lys Ala ArgCys Gly Ile Leu Ile Lys Glu Ala Lys Gln 145 150 155 160 Tyr Glu Glu AlaAla Lys Asn Ile Val Thr Ser Leu Asp Gln Phe Leu 165 170 175 His Gly AspGln Lys Lys Leu Glu Gly Val Ile Asn Ile Gln Lys Arg 180 185 190 Leu LysGlu Val Gln Thr Ala Leu Asn Gln Ala His Gly Glu Ser Ser 195 200 205 ProAla His Lys Glu Leu Leu Glu Lys Val Lys Asn Leu Lys Thr Thr 210 215 220Leu Glu Arg Thr Ile Lys Ala Glu Gln Asp Leu Glu Lys Lys Val Glu 225 230235 240 Tyr Ser Phe Leu Leu Gly Pro Leu Leu Gly Phe Val Val Tyr Glu Ile245 250 255 Leu Glu Asn Thr Ala Val Gln His Ile Lys Asn Gln Ile Asp GluIle 260 265 270 Lys Lys Gln Leu Asp Ser Ala Gln His Asp Leu Asp Arg AspVal Lys 275 280 285 Ile Ile Gly Met Leu Asn Ser Ile Asn Thr Asp Ile AspAsn Leu Tyr 290 295 300 Ser Gln Gly Gln Glu Ala Ile Lys Val Phe Gln LysLeu Gln Gly Ile 305 310 315 320 Trp Ala Thr Ile Gly Ala Gln Ile Glu AsnLeu Arg Thr Thr Ser Leu 325 330 335 Gln Glu Val Gln Asp Ser Asp Asp AlaAsp Glu Ile Gln Ile Glu Leu 340 345 350 Glu Asp Ala Ser Asp Ala Trp LeuVal Val Ala Gln Glu Ala Arg Asp 355 360 365 Phe Thr Leu Asn Ala Tyr SerThr Asn Ser Arg Gln Asn Leu Pro Ile 370 375 380 Asn Val Ile Ser Asp SerCys Asn Cys Ser Thr Thr Asn Met Thr Ser 385 390 395 400 Asn Gln Tyr SerAsn Pro Thr Thr Asn Met Thr Ser Asn Gln Tyr Met 405 410 415 Ile Ser HisGlu Tyr Thr Ser Leu Pro Asn Asn Phe Met Leu Ser Arg 420 425 430 7 1140DNA Bacillus thuringiensis 7 atgggtctga ttcacacaat caagctgaac tctaacaagaagtatggtcc tggcgatatg 60 actaacggga accagttcat catatccaag caagaatgggccacgattgg cgcatacatt 120 cagactggac tcggcttacc agtgaatgag caacagctgagaacccacgt taaccttagt 180 caagacatca gcataccatc tgacttttct caactctacgatgtgtattg ttctgacaag 240 actagtgcag aatggtggaa caagaatctc tatcctttgatcatcaagtc tgccaatgac 300 attgcttcat atggtttcaa agttgctggt gatccttcgatcaagaaaga tggttacttc 360 aagaagcttc aagatgaact cgacaacatt gttgacaacaactccgacga cgatgcgata 420 gccaaagcca tcaaggactt caaagcaaga tgtggcattctcatcaagga agccaagcag 480 tatgaagaag ctgccaagaa cattgtaaca tcattggatcagtttctcca tggagaccag 540 aagaagctcg agggtgtcat caacattcag aaacgtctgaaagaggttca aacagctctg 600 aatcaagccc atggggaatc cagtccagct cacaaagagcttcttgagaa agtgaagaac 660 ttgaagacca cacttgagag gaccatcaaa gctgaacaagacttggagaa gaaagtagag 720 tacagctttc tacttggacc cttgttaggc tttgttgtctacgagattct tgagaacact 780 gctgttcaac acatcaagaa tcaaatcgat gagatcaagaaacagttgga ttctgcgcaa 840 catgacttgg atcgcgatgt gaagatcatt ggaatgctcaacagcatcaa cactgacatt 900 gacaacttgt atagtcaagg acaagaagca atcaaagtctttcagaagct acaagggata 960 tgggccacta ttggagctca gatagagaat cttcgcaccacgtcccttca agaagtccaa 1020 gactctgatg atgctgatga gatacagatt gaacttgaggacgcatctga tgcatggtta 1080 gttgtggctc aagaagctcg tgacttcaca ctgaatgcctactcaaccaa cagtcgatag 1140 8 380 PRT Bacillus thuringiensis 8 Met GlyLeu Ile His Thr Ile Lys Leu Asn Ser Asn Lys Lys Tyr Gly 1 5 10 15 ProGly Asp Met Thr Asn Gly Asn Gln Phe Ile Ile Ser Lys Gln Glu 20 25 30 TrpAla Thr Ile Gly Ala Tyr Ile Gln Thr Gly Leu Gly Leu Pro Val 35 40 45 AsnGlu Gln Gln Leu Arg Thr His Val Asn Leu Ser Gln Asp Ile Ser 50 55 60 IlePro Ser Asp Phe Ser Gln Leu Tyr Asp Val Tyr Cys Ser Asp Lys 65 70 75 80Thr Ser Ala Glu Trp Trp Asn Lys Asn Leu Tyr Pro Leu Ile Ile Lys 85 90 95Ser Ala Asn Asp Ile Ala Ser Tyr Gly Phe Lys Val Ala Gly Asp Pro 100 105110 Ser Ile Lys Lys Asp Gly Tyr Phe Lys Lys Leu Gln Asp Glu Leu Asp 115120 125 Asn Ile Val Asp Asn Asn Ser Asp Asp Asp Ala Ile Ala Lys Ala Ile130 135 140 Lys Asp Phe Lys Ala Arg Cys Gly Ile Leu Ile Lys Glu Ala LysGln 145 150 155 160 Tyr Glu Glu Ala Ala Lys Asn Ile Val Thr Ser Leu AspGln Phe Leu 165 170 175 His Gly Asp Gln Lys Lys Leu Glu Gly Val Ile AsnIle Gln Lys Arg 180 185 190 Leu Lys Glu Val Gln Thr Ala Leu Asn Gln AlaHis Gly Glu Ser Ser 195 200 205 Pro Ala His Lys Glu Leu Leu Glu Lys ValLys Asn Leu Lys Thr Thr 210 215 220 Leu Glu Arg Thr Ile Lys Ala Glu GlnAsp Leu Glu Lys Lys Val Glu 225 230 235 240 Tyr Ser Phe Leu Leu Gly ProLeu Leu Gly Phe Val Val Tyr Glu Ile 245 250 255 Leu Glu Asn Thr Ala ValGln His Ile Lys Asn Gln Ile Asp Glu Ile 260 265 270 Lys Lys Gln Leu AspSer Ala Gln His Asp Leu Asp Arg Asp Val Lys 275 280 285 Ile Ile Gly MetLeu Asn Ser Ile Asn Thr Asp Ile Asp Asn Leu Tyr 290 295 300 Ser Gln GlyGln Glu Ala Ile Lys Val Phe Gln Lys Leu Gln Gly Ile 305 310 315 320 TrpAla Thr Ile Gly Ala Gln Ile Glu Asn Leu Arg Thr Thr Ser Leu 325 330 335Gln Glu Val Gln Asp Ser Asp Asp Ala Asp Glu Ile Gln Ile Glu Leu 340 345350 Glu Asp Ala Ser Asp Ala Trp Leu Val Val Ala Gln Glu Ala Arg Asp 355360 365 Phe Thr Leu Asn Ala Tyr Ser Thr Asn Ser Arg Met 370 375 380 91185 DNA Bacillus thuringiensis 9 atgattttag ggaatggaaa gactttaccaaagcatataa gattagctca tatttttgca 60 acacagaatt cttcagctaa gaaagacaatcctcttggac cagaggggat ggttactaaa 120 gacggtttta taatctctaa ggaagaatgggcatttgtgc aggcctatgt gactacaggc 180 actggtttac ctatcaatga cgatgagatgcgtagacatg ttgggttacc atcacgcatt 240 caaattcctg atgattttaa tcaattatataaggtttata atgaagataa acatttatgc 300 agttggtgga atggtttctt gtttccattagttcttaaaa cagctaatga tatttccgct 360 tacggattta aatgtgctgg aaagggtgccactaaaggat attatgaggt catgcaagac 420 gatgtagaaa atatttcaga taatggttatgataaagttg cacaagaaaa agcacataag 480 gatctgcagg cgcgttgtaa aatccttattaaggaggctg atcaatataa agctgcagcg 540 gatgatgttt caaaacattt aaacacatttcttaaaggcg gtcaagattc agatggcaat 600 gatgttattg gcgtagaggc tgttcaagtacaactagcac aagtaaaaga taatcttgat 660 ggcctatatg gcgacaaaag cccaagacatgaagagttac taaagaaagt agacgacctg 720 aaaaaagagt tggaagctgc tattaaagcagagaatgaat tagaaaagaa agtgaaaatg 780 agttttgctt taggaccatt acttggatttgttgtatatg aaatcttaga gctaactgcg 840 gtcaaaagta tacacaagaa agttgaggcactacaagccg agcttgacac tgctaatgat 900 gaactcgaca gagatgtaaa aatcttaggaatgatgaata gcattgacac tgatattgac 960 aacatgttag agcaaggtga gcaagctcttgttgtattta gaaaaattgc aggcatttgg 1020 agtgttataa gtcttaatat cggcaatcttcgagaaacat ctttaaaaga gatagaagaa 1080 gaaaatgatg acgatgcact gtatattgagcttggtgatg ccgctggtca atggaaagag 1140 atagccgagg aggcacaatc ctttgtactaaatgcttata ctcct 1185 10 208 PRT Bacillus thuringiensis 10 Met Ile LeuGly Asn Gly Lys Thr Leu Pro Lys His Ile Arg Leu Ala 1 5 10 15 His IlePhe Ala Thr Gln Asn Ser Ser Ala Lys Lys Asp Asn Pro Leu 20 25 30 Gly ProGlu Gly Met Val Thr Lys Asp Gly Phe Ile Ile Ser Lys Glu 35 40 45 Glu TrpAla Phe Val Gln Ala Tyr Val Thr Thr Gly Thr Gly Leu Pro 50 55 60 Ile AsnAsp Asp Glu Met Arg Arg His Val Gly Leu Pro Ser Arg Ile 65 70 75 80 GlnIle Pro Asp Asp Phe Asn Gln Leu Tyr Lys Val Tyr Asn Glu Asp 85 90 95 LysHis Leu Cys Ser Trp Trp Asn Gly Phe Leu Phe Pro Leu Val Leu 100 105 110Lys Thr Ala Asn Asp Ile Ser Ala Tyr Gly Phe Lys Cys Ala Gly Lys 115 120125 Gly Ala Thr Lys Gly Tyr Tyr Glu Val Met Gln Asp Asp Val Glu Asn 130135 140 Ile Ser Asp Asn Gly Tyr Asp Lys Val Ala Gln Glu Lys Ala His Lys145 150 155 160 Asp Leu Gln Ala Arg Cys Lys Ile Leu Ile Lys Glu Ala AspGln Tyr 165 170 175 Lys Ala Ala Ala Asp Asp Val Ser Lys His Leu Asn ThrPhe Leu Lys 180 185 190 Gly Gly Gln Asp Ser Asp Gly Asn Asp Val Ile GlyVal Glu Ala Val 195 200 205

1. An isolated, pesticidal protein wherein said protein comprises apesticidal fragment of the full-length Cry6A toxin of SEQ ID NO: 2,wherein said protein has a molecular weight between approximately 34 kDaand approximately 50 kDa.
 2. The protein of claim 1 wherein said proteinhas a molecular weight of approximately 40-48.7 kDa.
 3. The protein ofclaim 1 wherein said protein consists of a pesticidal fragment of thefull-length Cry6A toxin of SEQ ID NO:
 2. 4. The protein of claim 1wherein said protein comprises the amino acid sequence of SEQ ID NO: 6or a pesticidal fragment of SEQ ID NO:
 6. 5. The protein of claim 1wherein said protein consists of the amino acid sequence of SEQ ID NO: 6or a pesticidal fragment of SEQ ID NO:
 6. 6. The protein of claim 1wherein said protein comprises an amino acid segment of SEQ ID NO: 2from approximately amino acid 11 to approximately amino acid 443 of SEQID NO:
 2. 7. The protein of claim 1 wherein said protein consists of anamino acid segment of SEQ ID NO: 2 from approximately amino acid 11 toapproximately amino acid 443 of SEQ ID NO:
 2. 8. The protein of claim 1wherein said protein comprises the amino acid sequence of SEQ ID NO: 8.9. The protein of claim 1 wherein said protein consists of an amino acidsegment of SEQ ID NO: 2 from approximately amino acid 11 toapproximately amino acid 390 of SEQ ID NO:
 2. 10. A method ofcontrolling a coleopteran pest wherein said method comprises contactingsaid pest with an isolated, pesticidal protein wherein said proteincomprises a pesticidal fragment of the full-length Cry6A toxin of SEQ IDNO: 2, wherein said protein has a molecular weight between approximately34 kDa and approximately 50 kDa.
 11. The method of claim 10 wherein saidprotein has a molecular weight of approximately 40-48.7 kDa.
 12. Themethod of claim 10 wherein said protein consists of a pesticidalfragment of the full-length Cry6A toxin of SEQ ID NO:
 2. 13. The methodof claim 10 wherein said protein comprises the amino acid sequence ofSEQ ID NO: 6 or a pesticidal fragment of SEQ ID NO:
 6. 14. The method ofclaim 10 wherein said protein consists of the amino acid sequence of SEQID NO: 6 or a pesticidal fragment of SEQ ID NO:
 6. 15. The method ofclaim 10 wherein said protein comprises an amino acid segment of SEQ IDNO: 2 from approximately amino acid 11 to approximately amino acid 443of SEQ ID NO:
 2. 16. The method of claim 10 wherein said proteinconsists of an amino acid segment of SEQ ID NO: 2 from approximatelyamino acid 11 to approximately amino acid 443 of SEQ ID NO:
 2. 17. Themethod of claim 10 wherein said protein comprises the amino acidsequence of SEQ ID NO:
 8. 18. The method of claim 10 wherein saidprotein consists of an amino acid segment of SEQ ID NO: 2 fromapproximately amino acid 11 to approximately amino acid 390 of SEQ IDNO:
 2. 19. The method of claim 10 wherein said is produced by andpresent in a plant.
 20. An isolated polynucleotide that encodes aprotein of claim
 1. 21. A transgenic microbial or plant cell comprisinga polynucleotide of claim 20.